Inhibition of ADAMTS4 (aggrecanase-1) by tissue inhibitors of metalloproteinases (TIMP-1, 2, 3 and 4).

ADAMTS4 (aggrecanase-1) is considered to play a key role in the degradation of aggrecan in arthritides. The inhibitory activity of tissue inhibitors of metalloproteinases (TIMPs) to ADAMTS4 was examined in an assay using aggrecan substrate. Among the four TIMPs, TIMP-3 inhibited the activity most efficiently with an IC(50) value of 7.9 nM, which was at least 44-fold lower than that of TIMP-1 (350 nM) and TIMP-2 (420 nM) and >250-fold less than that of TIMP-4 (2 microM for 35% inhibition). These results suggest that TIMP-3 is a potent inhibitor against the aggrecanase activity of ADAMTS4 in vivo.

Acylated delphinidin 3-rutinoside-5-glucosides in the flowers of Petunia reitzii.

Two acylated anthocyanins were isolated from selected individuals of Petunia reitzii, and identified to be delphinidin 3-O-[6-O-(4-O-(4-O-(6-O-(trans-caffeoyl)-beta-D-glucopyranosyl)-tr ans-p-coumaroyl)-alpha-L-rhamnopyranosyl)-beta-D-glucopyranoside]- 5-O-[beta-D-glucopyranoside] and delphinidin 3-O-[6-O-(4-O-(4-O-(beta-D-glucopyranosyl)-trans-p-coumaroyl)-alph a-L-rhamnopyranosyl)-beta-D-glucopyranoside]-5-O-[beta-D-glucopyranoside ]. Nine known anthocyanins were also identified.

Differences in the floral anthocyanin content of red petunias and Petunia exserta.

In order to resolve a conflict between previous papers regarding the floral anthocyanins of red flowers of Petunia exserta, a naturally occurring species, the HPLC profile of this species was compared with that of commercial red garden petunias. Both HPLC profiles extremely superficially resemble each other in terms of relative amounts and retention times of the major anthocyanins. However, co-elution on HPLC of the mixed sample resulted in clear separation of the components. Three major anthocyanins in red petunias were determined to be cyanidin 3-sophoroside, cyanidin 3-glucoside and peonidin 3-glucoside, which exhibited similar behaviors on HPLC to delphinidin 3-glucoside. delphinidin-3-rutinoside and petunidin 3-rutinoside, respectively, the major floral anthocyanins of P. exserta.

Three groups of species in Petunia sensu Jussieu (Solanaceae) inferred from the intact seed.

The intact seed surface morphology in 45 taxa of Petunia sensu Jussieu native to South America (Petunia sensu Wijsman plus Calibrachoa) was compared under scanning electron microscopy. The existence of three groups of species, differentiated in terms of seed morphology, was revealed as follows: (1) all species of Petunia sensu Wijsman, having coarse wavy middle lamellae and anticlinal walls; (2) Calibrachoa parviflora and C. pygmaea, having fine wavy middle lamellae embedded in straight anticlinal walls; and (3) the other species of Calibrachoa, having straight middle lamellae and anticlinal walls. Close relationships between seed morphology and the other characteristics observable in the groups of species are discussed.

FGF-10 is a chemotactic factor for distal epithelial buds during lung development.

Fibroblast growth factor (FGF) signaling is required for normal epithelial branching in the respiratory system of several species. Recent studies have shown that FGF-10 may be a key regulator of lung branching morphogenesis, based on its pattern of expression in the early lung and its ability to induce epithelial budding in vitro. In this study we investigate whether FGF-10 is able to direct lung epithelial buds to proper positions during development . We maintained localized high levels of FGF-10 in cultured lungs using FGF-10-soaked heparin beads. FGF-10 exerts a powerful chemoattractant effect on the distal but not on proximal lung epithelium. Epithelial buds grow toward an FGF-10 source within 24 h, and subsequently form concentric layers of epithelium around the bead. BrdU incorporation analysis suggests that FGF-10, in contrast to FGF-7, is a modest proliferation factor for the lung epithelium. In the absence of mesenchyme FGF-10 requires an associated proliferative signal to induce bud migration. This can be provided by extract from lung mesenchyme, or by FGF-7, a growth factor also present in the early embryonic lung. FGF-10 does not seem to interfere with early epithelial cell differentiation. The chemoattractant effect of FGF-10 in the lung epithelium is reminiscent of the patterning effect of the Drosophila FGF ortholog branchless in the developing tracheal epithelium, suggesting that the function of these genes has been conserved during evolution.

Structure and expression of human fibroblast growth factor-10.

We isolated the cDNA encoding a novel member of the human fibroblast growth factor (FGF) family from the lung. The cDNA encodes a protein of 208 amino acids with high sequence homology (95.6%) to rat FGF-10, indicating that the protein is human FGF-10. Human FGF-10 as well as rat FGF-10 has a hydrophobic amino terminus ( approximately 40 amino acids), which may serve as a signal sequence. The apparent evolutionary relationships of human FGFs indicate that FGF-10 is closest to FGF-7. Chromosomal localization of the human FGF-10 gene was examined by in situ hybridization. The gene was found to map to the 5p12-p13 region. Human FGF-10 (amino acids 40 to 208 with a methionine residue at the amino terminus) was produced in Escherichia coli and purified from the cell lysate. Recombinant human FGF-10 (approximately 19 kDa) showed mitogenic activity for fetal rat keratinizing epidermal cells, but essentially no activity for NIH/3T3 cells, fibroblasts. The specificity of mitogenic activity of FGF-10 is similar to that of FGF-7 but distinct from that of bFGF. In structure and biological activity, FGF-10 is similar to FGF-7.

Use of ultrasonography in the detection of ureteric reflux in children suspected of having urinary infection.

The present study investigated whether ultrasonography was effective in detecting ureteric reflux in children suspected of having urinary infection. Seventeen children with febrile episodes and pyuria were enrolled. The ultrasound examination revealed ballooning of the renal pelvis during bladder contraction in 4 children, dilatation of the distal ureters in 6, and small kidney in 2. Cystography was performed on the 6 children with these ultrasound abnormalities and 1 child with two episodes of suspected urinary infection. Four children showed reflux. All of the 4 children had been found to have renal pelvic ballooning on ultrasound. None of the 10 children who did not undergo cystography had recurrence of urinary infection or significant bacteriuria during a median follow-up period of 12 months. Thus, scanning during bladder contraction was effective in detecting significant ureteric reflux.

Ultrasonography for the detection of ureteric reflux in infants with urinary infection.

Several less harmful methods than voiding cysto-urethrography for detecting significant ureteric reflux have been proposed. The present prospective study investigated whether ultrasonography was effective in identifying ureteric reflux in infants with their first febrile urinary infection. The subjects were 27 infants (24 boys and 3 girls) aged from 0 to 8 months. The urinary tract was scanned when the bladder was full, and before and during induced voiding. Infants with abnormal ultrasound findings underwent voiding cysto-urethrography. The other infants were followed and those who had a recurrence of urinary infection underwent voiding cystography. Ten children underwent cysto-urethrography, with eight refluxing ureters identified in six boys. Ultrasound revealed transient dilatation of the renal pelvis on voiding in five kidneys, transient dilatation of distal ureters in 12 and hydro-ureteronephrosis in two. Each of the five kidneys with pelvic dilatation on voiding was associated with ureteric reflux grades III or IV. Of the 17 children who did not undergo cysto-urethrography, only one had recurrence of urinary infection and was diagnosed with ureteric reflux. This girl was one of the three babies who were not scanned during voiding. More than half of the infants with febrile urinary infection were excluded from invasive examination without having recurrence of urinary infection. Thus, ultrasound scanning during voiding was effective for screening infants with their first urinary infection to detect significant ureteric reflux.

Problems of trace elements and vitamins during long-term total parenteral nutrition: a case report of idiopathic intestinal pseudo-obstruction.

An 8-year-old girl with chronic idiopathic intestinal pseudo-obstruction (CIIP), who is the first case of CIIP in Japan, has been receiving total parenteral nutrition (TPN) for more than 6 years. During this time, she experienced deficiencies of copper, zinc, vitamin A, vitamin B12, folic acid, and biotin, and an excess of vitamin A; she exhibited a series of signs and symptoms due to these deficiencies and vitamin A overdosage. Nevertheless, careful monitoring of serum levels of trace elements and vitamins and appropriate therapy have almost solved these problems. She has achieved normal physical and mental development and goes to school, while receiving home parenteral nutrition with an ambulatory infusion system.

Antibody-dependent cell-mediated cytotoxicity against influenza virus-infected cells.

The three major immunocompetent cells in human peripheral blood (lymphocytes, neutrophils, and monocytes) were shown to be effector cells for antibody-dependent cell-mediated cytotoxicity (ADCC) against influenza virus-infected baby hamster kidney cells in vitro. Lymphocyte cytotoxicity was mediated by FcIgG receptor-bearing null cells and T gamma cells. These effector populations were best defined by HNK-1, a monoclonal antibody to human natural killer and ADCC-mediator cells. Antibody responsible for ADCC against influenza virus-infected cells was detectable in sera of young children after natural infection and after vaccination with inactivated and live attenuated viruses. ADCC antibody appeared before hemagglutination-inhibiting antibody and persisted for at least one year after vaccination with live attenuated vaccine. ADCC antibody was subtype-specific but quite broadly reactive within a subtype. Both hemagglutinin and neuraminidase were antigenic determinants for ADCC antibody. An anamnestic response to the original strain was observed after challenge with influenza virus of a heterologous subtype.

Ability of human cord blood lymphocytes to mediate antibody-dependent cellular cytotoxicity against influenza virus-infected cells.

Cord blood lymphocytes, monocytes, and neutrophils from newborns were shown to mediate antibody-dependent cellular cytotoxicity (ADCC) against influenza virus-infected cells. Antibody mediating ADCC was detectable in cord plasma, indicating that all components necessary for ADCC against influenza virus-infected cells are present in newborns. Among adult lymphocytes, two effector cell populations of influenza ADCC are recognized: non-T and T gamma cells. Each of these cell types expresses an antigen recognized by monoclonal HNK-1 antibody. The proportion of HNK-1 antigen-positive lymphocytes in cord blood was markedly lower than in adult blood; furthermore, ADCC was mediated by cord blood lymphocytes which were HNK-1 negative. By lymphocyte fractionation, the effector lymphocytes in cord blood were, as in adults, non-T and T gamma cells, suggesting that HNK-1 antigen is not expressed on these cell lineages in newborns.

Characterization of the human peripheral blood effector cells mediating antibody-dependent cell-mediated cytotoxicity against respiratory syncytial virus.

Peripheral blood lymphocytes were separated into several subpopulations and evaluated for their ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) against respiratory syncytial virus (RSV)-infected HeLa cells. Using erythrocyte rosetting methods, nylon wool filtration, and cytolysis with OKT-3 monoclonal antibody, two lymphocyte subpopulations were shown to mediate RSV-ADCC; non-T, non-B, and IgG-Fc receptor-bearing lymphocytes and E-rosetting cells with IgGFc receptors (T gamma cells). Removal of phagocytic cells did not alter ADCC activity. Monoclonal antibody to human NK and K cells, HNK-1, recognized these two lymphocyte effector subpopulations.

Conformational-energy studies of tetrapeptide opiates. Candidate active and inactive conformations.

The conformational behavior of four tetrapeptide enkephalin analogues (Tyr-Gly-Gly-Phe-OH, Tyr-Gly-Gly-Phe-NH2, Tyr-D-Ala-Gly-Phe-NH2, and Tyr-D-Ala-Gly-(NMe)Phe-NH2) was examined to identify conformations that are active and inactive at the opiate analgesic receptor. By using an empirical energy program, conformational energies were obtained for the optimized geometries of each tetrapeptide. Two methods of selecting candidate active conformations from low-energy conformers were used. In the first method, inactive conformers were designated as low-energy conformations of the very weak tetrapeptide, Tyr-Gly-Gly-Phe-OH. These candidate inactive conformers had geometries resembling beta V, beta I, "random" peptide conformations. Candidate active conformers selected were low-energy conformations found for both Tyr-D-Ala-Gly-Phe-NH2 and Tyr-D-Ala-Gly-(NMe)Phe-NH2 but not low-energy conformers for Tyr-Gly-Gly-Phe-OH. In the second method of selection, conformers with relative energies in the active and inactive peptides that followed the potency order Tyr-Gly-Gly-Phe-OH much less than Tyr-D-Ala-Gly-Phe-NH2 less than or equal to Tyr-D-Ala-Gly-(NMe)Phe-NH2 were chosen as candidate active conformers. By using both methods of selection, a beta II' bend geometry was found as the active conformer. This beta II' conformer was not stabilized by a 1-4 hydrogen bond, but instead was stabilized by a hydrogen bond between the tyrosine amine hydrogen atom and the phenylalanine carbonyl oxygen atom. The effect of C-terminal amide derivitization on peptide conformation was also examined by comparing the conformational profiles of Tyr-Gly-Gly-Phe-OH and Tyr-D-Ala-Gly-Phe-OH with their amides Tyr-Gly-Gly-Phe-OH-NH2 and Tyr-D-Ala-Gly-Phe-NH2. No significant difference in conformational behavior was found for the Tyr-Gly-Gly-Phe pair; however, a difference in conformational behavior was found between the Tyr-D-Ala-Gly-Phe acid and amide. Thus, on the basis of conformational data, the Tyr-Gly-Gly-Phe-NH2 analogue is predicted to have very weak opiate activity.

[Clinical evaluation of cefsulodin in Pseudomonas infections in children].

Cefsulodin (CFS) was evaluated for its safety and efficacy in 14 children with Pseudomonas aeruginosa infections. The diagnoses included pneumonia (4), sepsis (1), presumed sepsis (4), acute postoperative ascending cholangitis (1), acute postoperative peritonitis with wandering pneumonia (1), acute enterocolitis with acute UTI (1), recurrent UTI (1), and acute cystitis (1). CFS was administered intravenously with a daily dose of 93 to 299 mg/kg in the cases with normal renal functions. CFS was effective in all but one case both clinically and bacteriologically. A case of pneumonia whose isolate was resistant to CFS responded poorly. Mild transient eosinophilia was observed in 3 cases, but no severe adverse reactions were encountered. Peak MIC values of 18 clinical isolates of P. aeruginosa were 1.56 mcg/ml, 0.39 to 0.78 mcg/ml and 12.5 mcg/ml for CFS, gentamicin, and sulbenicillin, respectively. A half life of the serum CFS levels was 1.09 hours after intravenous bolus injection of 20 to 25 mg/kg of CFS (n = 2). A cerebrospinal-fluid level and biliary levels measured in cases with inflamed meninges or with cholangitis were well above the MIC value. From the present study, CFS appeared to be a safe and effective antibiotic when used in children with susceptible Pseudomonas infections. Combined use of another antibiotic should be considered in the case with polymicrobial infections because of the CFS's very narrow spectrum.

[Clinical evaluation of cefoxitin in children (author's transl)].

Cefoxitin (CFX) was evaluated for its safety and efficacy in children. Fifteen patients were treated with 73-125 mg/kg per day of CFX by intravenous administrations. The diagnosis of the patients were acute pharyngitis (4), pneumonia (2), pertussis and pneumonia (1), urinary tract infection (3); and the remaining 5 patients were esteemed to have nonbacterial infections. All the 10 patients of bacterial infections were cured after the CFX therapy. The pathogens recovered were Streptococcus pyogenes (1), Streptococcus pneumoniae (3), Haemophilus influenzae (2), Escherichia coli (2), enteropathogenic Escherichia coli (1), and Klebsiella pneumoniae (1). All the strains isolated were susceptible to CFX, but the 2 isolates of Haemophilus influenzae had relatively high MIC values (12.5 mcg/ml). Diarrhea (3 cases) and transient neutropenia (1 case) were found to be associated with the CFX therapy. However, no severe adverse reactions were encountered. Half-life of the serum level was short (24.1 minutes) and excretion into the urine was rapid. CSF concentration obtained 30 minutes after an intravenous injection of 50 mg/kg of CFX in 1 case with inflamed meninges was considerably high (8.3 mcg/ml). CFX appears to be a safe and effective antibiotic when used in children with susceptible bacterial infections.

Quantum chemical calculations on the two-step mechanism of proflavin binding to DNA.

Quantum chemical calculations on the binding of proflavin to DNA lead to a model in which the outside binding to a phosphate group leads to an induced fit in the intercalation receptor site. The calculations suggest hydrogen bonding of the amine groups of the outside bound proflavin to the anionic oxygen of the backbone phosphate. The resulting partial neutralization facilitates the conformational transitions required for intercalation. The results are consistent with the observed preference of proflavin for dCpdG over dGpdC sequences and with the observed kinetics of the binding reaction.